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Journal of Virology, December 2000, p. 11663-11670, Vol. 74, No. 24
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

A Functional NSP4 Enterotoxin Peptide Secreted from Rotavirus-Infected Cells

Mingdong Zhang,1,dagger Carl Q.-Y. Zeng,1 Andrew P. Morris,2 and Mary K. Estes1,*

Division of Molecular Virology, Baylor College of Medicine,1 and Department of Pharmacology, Physiology, and Integrative Biology, University of Texas Health Science Center,2 Houston, Texas 77030

Received 27 March 2000/Accepted 3 October 2000

Previous studies have shown that the nonstructural glycoprotein NSP4 plays a role in rotavirus pathogenesis by functioning as an enterotoxin. One prediction of the mechanism of action of this enterotoxin was that it is secreted from virus-infected cells. In this study, the media of cultured (i) insect cells infected with a recombinant baculovirus expressing NSP4, (ii) monkey kidney (MA104) cells infected with the simian (SA11) or porcine attenuated (OSU-a) rotavirus, and (iii) human intestinal (HT29) cells infected with SA11 were examined to determine if NSP4 was detectable. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis-Western blotting, immunoprecipitation and N-terminal amino acid sequencing identified, in the early media from virus-infected cells, a secreted, cleavage product of NSP4 with an apparent molecular weight of 7,000 that represented amino acids 112 to 175 (NSP4 aa112-175). The secretion of NSP4 aa112-175 was not affected by treatment of cells with brefeldin A but was abolished by treatment with nocodazole and cytochalasin D, indicating that secretion of this protein occurs via a nonclassical, Golgi apparatus-independent mechanism that utilizes the microtubule and actin microfilament network. A partial gene fragment coding for NSP4 aa112-175 was cloned and expressed using the baculovirus-insect cell system. Purified NSP4 aa112-175 increased intracellular calcium mobilization in intestinal cells when added exogenously, and in insect cells when expressed endogenously, similarly to full-length NSP4. NSP4 aa112-175 caused diarrhea in neonatal mice, as did full-length NSP4. These results indicate that NSP4 aa112-175 is a functional NSP4 enterotoxin peptide secreted from rotavirus-infected cells.

* Corresponding author. Mailing address: Division of Molecular Virology, Mail Stop BCM-385, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030-3411. Phone: (713) 798-3585. Fax: (713) 798-3586. E-mail: mestes{at}bcm.tmc.edu.

dagger Present address: Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.

Journal of Virology, December 2000, p. 11663-11670, Vol. 74, No. 24
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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